Prevalence of common autosomal recessive mutation carriers in women in the Southern Vietnam following the application of expanded carrier screening

The common autosomal recessive (AR) mutation carrier is still unknown in Vietnam. This study aims to identify the most common AR gene mutation carriers in women of reproductive age to build a Vietnamese-specific carrier screening panel for AR and X-linked disorders in the preconception and prenatal healthcare program. A cross-sectional study was conducted at University Medical Center–Branch 2 in Ho Chi Minh City from December 1st, 2020, to June 30th, 2023. 338 women have consented to take a 5 mL blood test to identify 540 recessive genes. The carrier screening panel was designed based on the American College of Medical Genetics and Genomics (ACMG)-recommended genes and suggestions from 104 clinical experts in Vietnam. Obstetricians and genetic experts counseled all positive testing results to discuss the possibility of recessive diseases in their offspring. The most common recessive disorders were defined at a prevalence of 1 in 60 or greater, and those were added to a Vietnamese-specific carrier screening panel. 338 non-pregnant and pregnant women underwent the expanded carrier screening (ECS). The carrier frequency was 63.6%, in which 215 women carried at least one AR gene mutation. GJB2 hearing impairment was identified as the most common chronic condition (1 in 5). The second most common AR disorder was beta-thalassemia (1 in 16), followed by cystic fibrosis (1 in 23), G6PD deficiency (1 in 28), Wilson’s disease (1 in 31), Usher’s syndrome (1 in 31), and glycogen storage disease (1 in 56). Seven common recessive genes were added in ethnic-based carrier screening. Women in the South of Vietnam have been carried for many recessive conditions at high frequency, such as hearing impairment, genetic anemia, and cystic fibrosis. It is necessary to implement a preconception and prenatal screening program by using seven widely popular AR genes in a Vietnamese-specific carrier screening panel to reduce the burden related to AR and X-linked disorders.

more recessive conditions in high-risk populations at a low cost.At the same time, an ECS increases the detection rate of the condition, even in minority groups, without causing stigmatization in the community 1 .According to Rowe et al., the more AR gene mutations are screened, the more carriers will be detected 1 .However, ECSs are highly expensive and usually give out unrealistic possibilities when implemented in low carrier frequencies.Therefore, knowing the common recessive gene panel becomes an essential foundation for setting up a specific carrier screening program in the population (Fig. 1).
To our knowledge, ECS was recommended by specialists in the European Society of Human Genetics (ESHG) in 2016 and the American College of Obstetricians and Gynecology (ACOG) in 2017 with specific guidelines that demonstrated the feasibility of applying for pre-pregnancy and prenatal screening, capacity to link with newborn screening program and establishment of a suitable gene panel based on clinical evidence of severe childhood-onset diseases with the frequency of 1 in 100 or greater 4 .In terms of clinical practices, each country has designed specific panel screening based on ethnicity, popular AR disorders, and acceptability of individuals.In the UK, a preconception and prenatal carrier screening panel has been established since 2017 to early diagnose genetic anemias (beta-thalassemia, sickle cell anemia), Tay-Sachs disease, and cystic fibrosis 5 .In China, Shi et al. applied in 2021 an ECS panel with 11 conditions, including dystrophinopathies (Duchenne muscular dystrophy and Becker muscular dystrophy), anemias (alpha-thalassemia and beta-thalassemia), autosomal recessive deafness (DFNB4), phenylketonuria (PKU), glycogen storage disease type II, hemophilia A and B, and spinal muscular atrophy (SMA) 6 .His study recruited 1321 Chinese Hong Kong women who underwent the ECS and reported the carrier frequency at 19.23% 6 .Fu et al. also reported in China that 66.9% of participants preferred preconception recessive carrier screening for deafness 7 .In Australia, Beard et al. conducted an ECS in 10 women (8 pregnant women and 2 women of reproductive age) for cystic fibrosis (CF), spinal muscular atrophy (SMA), and fragile X syndrome (FXS).The results showed that all of them supported this screening positively and suggested doing this test before preconception 8 .In the Netherlands, Mathijssen et al. found that 97% of participants who underwent a preconception and antenatal carrier screening test for 4 recessive disorders (PCH2, FADS, rhizomelic chondrodysplasia punctata type 1, and osteogenesis imperfecta) wanted to recommend it to others 9 .Those studies have proved the necessity and feasibility of designing an ethnic-specific-based carrier screening panel to screen AR and X-linked diseases.
In Vietnam, the public perception of AR carriers is still unaware in reproductive healthcare due to a lack of recessive condition data.Although AR and X-linked disorders have led to up to 20% of neonatal mortality and accounted for 18% of infant hospitalization due to chronic disability (Ministry of Health, Vietnam, 2018, http:// www.moh.gov.vn), an official Vietnamese-specific carrier screening panel in Vietnam has not been established yet.The newborn screening program for Glucose-6-phosphate dehydrogenase (G6PD) deficiency and congenital hypothyroidism has been implemented in several maternity hospitals but hasn't significantly impacted public health.Whereas the prenatal screening program for inherited anemia, especially beta-thalassemia, which has been applied since 2013 10 , showed a high prevalence of thalassemia carriers in Vietnamese pregnant women (6.4% for alpha-thalassemia and 1.32% for beta-thalassemia).This early diagnosis allows their children to gain more opportunities to have premature and effective treatment (bone marrow transplantation).Recently, Tran et al. conducted a cohort study in 985 Vietnamese women and men to evaluate autosomal dominant and recessive mutation genes in the ECS panel with 118 AR conditions.The results showed 7 popular recessive gene mutations, including GJB2 (deafness), VPS13B (Cohen syndrome), HBB (beta-thalassemia), SLC25A13 (Citrin deficiency), GCNT2 (cataract 13), TMEM67 (Joubert syndrome), PAH (Phenylketonuria) 11 .Facing a very high AR carrier prevalence in Vietnam, the authors suggested a larger panel-based has been recommended to build up an optimal ECS in the Vietnamese population 11 .
Responding to the urgent need to improve the quality of reproductive healthcare programs, including preconception and prenatal counseling for AR and X-linked disorders in Vietnam, we conducted this study to identify the prevalence of most common recessive conditions in all non-pregnant and pregnant women coming to University Medical Center-Branch 2 in Ho Chi Minh city.Following the results, a comprehensive and specific carrier screening panel will be designed and implemented in Vietnam's preconception and prenatal care programs.

Results
338 Vietnamese women who accepted ECS were recruited at University Medical Center-Branch 2 in Ho Chi Minh City from December 1st, 2020, to June 30th, 2023.The mean age of participants was 27.3 ± 6.5 years.25 of them were pregnant with gestational age at 12 +3 weeks of gestation.Most (73%) live in urban areas in the South of Vietnam.The family genetic histories had shown unremarkable notice in these women.540 genes for AR and X-linked disorders were analyzed in 338 non-pregnant and pregnant women.The carrier prevalence for at least one recessive disease was 63.6% (215/338), and detailed carrier frequencies of recessive conditions are presented in Table 1.Among 215 women carriers, 134 individuals (63.6%) were carried for one recessive disease, and one woman (0.3%) had as many as 5 recessive diseases (Fig. 2).Only one pregnant woman carried 2 recessive genes (GJB2 and ATP7B, both were found in heterozygous and pathogenic type) at 7 +1 weeks, and 11 of pregnant women carried only one recessive gene (GJB2, G6PD, CFTR).All pregnant women carrying recessive genes followed the special prenatal care for high-risk pregnancies.
For female carriers who were married, their partners were invited to perform ECS.Unfortunately, only one male accepted this test and got a negative result.Four non-pregnant women carried GJB2 with c.109G>A (p.Val37Ile) variant found in the pathogenic homozygous type.These four women were of reproductive age from 22 to 34 years old.One woman has married with gravida 1 and parity 1.Her son is 4 years old and healthy.Her husband was accepted to be tested and presented negative for GJB2 mutation.Three remaining women were single.All four women declared that they had no health issues before performing the ECS test and weren't aware of hearing impairment.After receiving the positive test, they underwent an auditory check and were confirmed a mild hearing deficiency by an otolaryngologist.Three single women received a post-test consultation and understood that it is required to have the partner's GJB2 test to evaluate the risk of hearing impairment in their future babies.
The second common recessive gene was HBB (6.2%), mainly in heterozygous and pathogenic types.The c.79G>A (p.Glu27Lys) variant was the popular variant (85.7%, 18/21).No pregnant women carrying the HBB gene were found in the present study.
CFTR was identified as the third common recessive carrier in our study at 4.4%, in which c.1210-11T>G found 80% (12/15) in heterozygous and pathogenic type.Two pregnant women were detected carrying CFTR at 7 +0 and 7 +4 weeks of gestation.Those pregnancies were followed at the high-risk pregnancy department until giving birth.
The fourth prevalence of recessive condition was G6PD deficiency coded by the G6PD gene with the carrier frequency 3.6% and c.961G>A (p.Val321Met) often observed in heterozygous and pathogenic type (42.7%, 5/12).
The fifth and sixth most common AR conditions were Wilson's disease and Usher syndrome, determined by ATP7B and USH2A.Its carrier frequency was the same rate of 3.3%.The ATP7B c.2459C>T (p.Thr850Ile) variants were defined as likely to be the possible pathogenic type (45.5%, 5/11).No pregnant woman was detected carrying ATP7B in this study.For the USH2A variant, c.6969C>T (p.Thr2310Met) was a popular likely pathogenic type (27.3%, 3/11) illustrated in Table 2.
The seventh most popular recessive gene was GAA , with a prevalence of 1.8%.The dominant variant was c.761C>T (p.Ser254Leu) in heterozygous and pathogenic type (40%, 2/5) shown in Table 2.
The rest of the common AR conditions were detected at 1.2% and 1.5%.Those chronic disorders were related to metabolic disorders and congenital anomalies coded by AGTX, GALC, GRHPR, RDH12, and RPGRIP1L (Table 2).

The prevalence of AR carrier in Vietnam
Analyzing 540 genes based on ACMG-recommended genes and 104 Vietnamese experts' suggestions in 338 Vietnamese women, we found the carrier prevalence for at least one AR or X-linked condition was 63.6% (Fig. 1).This result was distinguished from previous studies due to differences in popular demographics, sample size, and the applied ECS gene panel.In the USA, Punj et al. identified 78% of carriers with at least one positive variant after performing an ECS testing in 728 genes for medically actionable conditions in 131 women and 71 of their  14 .Obviously, the more AR gene mutations are accounted for, the more carriers will be detected 1 .63.6% of Vietnamese women carried at least one AR variant, which is quite prevalent.The finding has illustrated the actual context of high-risk carriers for AR and X-linked disorders that need to bring more actions to improve the quality of reproductive healthcare in Vietnam.Therefore, the establishment of a preconception and prenatal carrier screening panel is a righteous choice since it could detect Vietnamese-specific AR and X-linked conditions at an affordable cost.

The most popular AR carriers
Understanding carrier frequency in the Southeast Asian population is still unclear due to limited data from genetic carrier screening in these countries.According to Lazarin et al., the carrier frequency in East Asians was generally lower than in Ashkenazi Jews and Caucasians (8.5% versus 43.6% and 32.6%) 15 .He also highlighted that GJB2-related hearing loss was the most common AR disorder (1 in 22), followed by beta-thalassemia disease (1 in 78) and SMA (1 in 85) 15 .
In the present study in Vietnam, 215 women were detected to carry at least one AR condition associated with 165 pathogenic and 124 likely pathogenic variants.These were similar to those of Chetruengchai et al. in Thailand (640 individuals carried 172 pathogenic and probably pathogenic variants) 14 .Considering the AR and X-linked condition with a frequency greater than 1.7% (1 in 60) as the most common disorders, we obtained seven top-carried genes in Vietnamese women, illustrated in Table 1.Generally, we could divide the common AR and X-linked conditions into 4 groups to discuss: the group of hearing impairment contained GJB2 and USH2A; the group of genetic anemia including HBB and G6PD genes; the group of cystic fibrosis (CFTR); the group of metabolism disorders coded by ATP7B and GAA gene.
With higher rates of the most common recessive genes and a larger spectrum of AR and X-linked disorders, our results differed significantly from findings in Thailand and China.According to Chetruengchai et al., the top gene carrier frequency was HBB (19.6%), followed by G6PD (7.7%), then HBA2 (3.96%) 14 .Besides, Shi et al. also indicated the first two common recessive genes were HBA1/HBA2 and HBB (7.76% and 2.25%, respectively), and the next two common were PAH for phenylketonuria and SLC26A4 for autosomal recessive deafness 4 (2.18% and 2.03%) 6 .Even when these three studies were conducted in the same Southeast and East Asian region, the AR and X-linked carriers presented differently due to different distributions of ethnicity, choices of screening methods, and the cutoff settings for the screening program.This would affirm the importance of building a Vietnamese-specific panel in the carrier screening program.
Compared to another study in Vietnam, our result was consistent with those reported by Tran et al., who also identified the highest recessive gene carrier frequency was GJB2 (1 in 6), and the third common was HBB (1 in 23) 11 .Both studies provided strong evidence to take genes GJB2 and HBB into a Vietnamese-specific carrier screening panel to detect early hearing impairment and inherited anemia in the preconception and prenatal care program.
Regarding other AR conditions recommended by Guo and Gregg in South and East Asia, and ACMG such as autoimmune polyendocrinopathy syndrome type 1, Usher's syndrome type IIa, SMA, and fragile X syndrome 4,16,17 , those were not found or found 1% or below (1 in 100), so we did not take into our discussion.

Hearing loss disorder (GJB2 and USH2A)
Focused on the GJB2-related hearing loss gene, the c.109G>A (p.Val37Ile) variant was determined as the most prevalent pathogenic variant (85.1%) in Vietnam.This result was distinguished from those of Wattanasirichaigoon et al. in Thai reported p.V37I variant at 11.1% 18 , and Tsukada et al. in Japan identified c.235delC (p.L79fs) variant at 49.8% 19 .Each ethnic individual indeed provided specific pathogenic variants.The diversity of variant data obtained from different studies in South and East Asia has supported the design of an ethnic-based carrier screening program and enriched the landscape of pathogenic gene panels worldwide.We can confidently add GJB2 with c.109G>A (p.Val37Ile) variant to the Vietnamese-specific ECS panel.
In literature, p.Val37Ile was often associated with bilateral mild to moderate hearing loss and adult-onset phenotype 20 .Nevertheless, it should be more cautious to explain to carrier individuals about their risks in preconception and prenatal counseling.In our study, among 57 women carrying pathogenic c.109G>A, four of them were found to be homozygotes and immediately underwent post-test counseling to perform auditory testing.An otolaryngologist confirmed their diagnosis as mild hearing loss, and they received a detailed followup auditory check for themselves and their offspring.Identifying GJB2-related hearing loss enabled women in our cohort to be aware of their status, either carried or suffered from chronic disability, and realize their risk of having offspring with AR conditions.
Besides GJB2-related hearing loss, we also identified 3.3% (1 in 31) of women carrying the USH2A gene that caused Usher syndrome, characterized by moderate to severe deafness at birth and progressive retinitis pigmentation 21 .The carrier frequency of Usher's syndrome was determined at around 1 in 70 in most ethnic populations, according to Koenekoop et al. 22 .However, USH2A and Usher syndrome have not been documented in Vietnam.A USH2A carrier frequency of 1 in 31 in our study was even two and half times higher than the previous study that highlighted Vietnamese women at high risk of hearing impairment and vision issues.That's why bringing GJB2 and USH2A genes into the ECS panel in Vietnam is reasonable.

Genetic anemia (HBB and G6PD deficiency)
Beta-thalassemia, a genetic anemia leading to life-threatening outcomes, was the second most common AR disorder (6%, 1 in 16) in the present study.Our report was quite consistent with those identified by Tran et al. in 2021 in Vietnam (1 in 23) 11 but was distinguished from Shi et al., who found the most common AR condition was alpha-thalassemia with 7.8% (n = 103) through conducting a "small-size" ECS panel containing 11 AR and X-linked disorders in 1321 Chinese pregnant women 6 .This difference emphasized the ethnic-specific genes coded AR diseases and research participants.
Since the national program of genetic anemia screening has been widely implemented in Vietnam, what we obtained in this cohort provided positive support for actual target carrier screening for hereditary anemia in preconception and prenatal care.Indeed, the national genetic anemia carrier screening program not only enhanced the awareness and knowledge of this reduced life span condition but also detected 61.85% of fetuses carried alpha-thalassemia, 13.07%carried beta-thalassemia, and 0.61% carried others (HbE disease, alphathalassemia/HbE, and beta-thalassemia/HbE) 23 .Following this target carrier screening program, newborns can get an immediately detailed treatment and improve their quality of life.
G6PD deficiency, an inherited X-linked disorder, is known as a significant risk factor for hemolytic anemia in many Southeast Asians.In Thailand, Sathupak et al. found its prevalence was 19.8% (127/640) 24 , and Chetruengchai et al. determined the carrier rate of the G6PD gene was the third common at 3.4% (55/1642) 14 .Realizing early-onset anemia in newborns and its consequences, the newborn G6PD screening program has been conducted in several maternity centers since 2010 in Vietnam.Nevertheless, this postnatal screening program did not demonstrate a remarkable impact to improve genetic hemolytic anemia.The frequency of G6PD deficiency was reported to still be high in southern Vietnam (8.7% in ethnic Kinh and 14% in ethnic S'tieng), with the most common variant called G6PD Viangchan 25 .The G6PD gene carrier frequency in the present study was 3.6% (1 in 28), even lower than those reported by Hue et al. 25 , demonstrating the Vietnamese population is at high risk of hemolytic anemia.G6PD should be added to the Vietnamese-specific ECS panel to provide early diagnosis and treatment for this X-linked disorder in reproductive women and their partners.

Cystic fibrosis
Cystic fibrosis (CF), an inherited disorder causing persistent lung infections and pancreatic insufficiency, has been seen as more common in individuals of Ashkenazi Jewish and Caucasian than other ethnicities, such as the African and Asian populations 4 .Since this condition is carried throughout individuals' life spans, ACOG and ACMG recommended screening the CF gene in all women considering pregnancy 4,16 .
Unlike thalassemia awareness, the CF condition and its data had not been well documented in Vietnam.Additionally, no CF carrier screening is conducted in preconception and prenatal care.Herein, we had the CF carrier frequency (4.4%, 1 in 23), which kept the third common prevalence in the present study, and that was the first CF carrier screening report in Vietnamese women of reproductive age.
1 in 23 carried CFTR showed significantly close to the Ashkenazi Jewish (1 in 24) and Non-Hispanic White (1 in 25), considered a high-risk group of CF carriers.We wondered if the Vietnamese population is also a high-risk carrier of this AR disease.Recently, Vaidyanathan S et al. suggested that CF condition has become more common in South Asians with specific CFTR variants distinguished from non-Hispanic Whites 26 .Indeed, c.1210-11T>G pathogenic variant found 80% (12/15) can be determined as a specific CFTR variant of CF carriers in Vietnam.Regarding preconception and prenatal counseling, CF conditions can be effectively prevented if the affected individuals, including carriers and their offspring, are identified and intervened early.Two pregnant women at early gestational age (< 7 weeks) carried CFTR in our study.Both received post-test counseling to understand the risk of their child and scheduled a detailed scanning at 18 weeks of gestation to evaluate fetal soft markers to diagnose the possibility of postnatal CF early.Our finding facilitated the broadening of the genetic landscape of CFTR and added this gene to the Vietnamese-specific ECS panel.

Metabolism disorders (ATP7B and GAA )
Interestingly, we also discovered two more common genes related to AR metabolism disorders that had not been mentioned in Vietnam yet.The first AR metabolism disease detected is Wilson's disease, a disorder of copper metabolism coded by the ATP7B gene.Wilson's disease has been considered the most common AR condition in Korea, with the carrier frequency found to be 1 in 53 27 , but its prevalence was nearly two times lower than our result (1 in 31, 3.3%).Recently, Yamaguchi et al. found the carrier frequency of this condition in Japan was 2.2%-2.8%,which was still lower than our findings 28 .Wilson's disease was one of the most common recessive conditions in Vietnam, and it needed to bring this gene was found in the Vietnamese ECS panel.
The second AR metabolism condition is glycogen storage disease type II, also called Pompe disease, another AR disorder caused by deficiency of the lysosomal alpha-glucosidase.This condition is characterized by an accumulation of glycogen within the lysosomes, causing progressive destruction of cardiac and skeletal muscle 29 .The sooner the diagnosis of this condition is made, the more the newborn could benefit from earlier enzyme replacement treatment 30 .Even though Pompe disease is found to be the most common disease in East Asia (1 in 77, 1.3%) 30 , this data has not been reported in Vietnam.In our cohort, GAA carrier frequency was found to be 1 in 56 (1.8%), which was one and a half times higher than the overall population, according to Park's study 30 .It affirmed the feasibility of building an ECS panel with the GAA gene.

Conclusions
In summary, genetic carrier testing has not been familiar to Vietnamese women in preconception and prenatal counseling because of the high-cost expense and unawareness of popular AR and X-linked diseases.Hence, our obtained results are considered the first essential foundation to confirm the benefit of genetic carrier screening and better understand the Vietnamese recessive genetic landscape.Based on current research, a Vietnamesespecific ECS panel for the seven most common recessive conditions was built from GJB2, USH2A, HBB, G6PD, CFTR, ATP7B, and GAA .This Vietnamese-specific ECS panel has responded to the actual clinical needs, matched with cost-effectiveness, and facilitated the provision of better genetic counseling in preconception and prenatal care in Vietnam.

Study methods and population
This cross-sectional study was conducted from December 1st, 2020, to June 30th, 2023.We collected data from non-pregnant women and pregnant women from 18 to 40 years old when they came to the Department of Obstetrics and Gynecology, University Medical Center-Branch 2 in Ho Chi Minh City.This study was conducted in a single medical center, University Medical Center-Branch 2, in Ho Chi Minh City.A total of 338 women were recruited in this study after giving their consent to participate.
3 obstetricians performed a pre-test counseling to collect personal information and explain the research procedure and benefits of ECS for 540 genes by using next-generation sequencing.Next, all women were taken a sample of 5 mL of blood in the peripheral vein kept in an EDTA tube.Then, these research samples were sent to the Molecular Biology Center to analyze 540 genes.Tran et al. reported a high prevalence of AR carriers in the Vietnamese population 11 .Hence, the cut-off of carrier frequency was defined as 1.7% (1 in 60) or greater, following the top popular list of AR gene mutations detected in this study.All recessive disorders with a prevalence greater than 1 in 60 are considered common diseases, and their genes will be added to the Vietnamese-specific carrier screening panel.
All results were given back to participants via both postal mail and email.Women detected carrying at least one recessive disease received post-testing counseling from 3 obstetricians and 1 genetic expert.Their partners were also invited to have ECS for 540 genes to predict the possibility of suffering from AR conditions in their children.

ECS panel design for 540 recessive genes
Since the conception of carrier screening and the data of most common AR and X-linked conditions are very new and still unknown in Vietnam, we conducted a survey to collect the list of AR and X-linked disorders in 104 Vietnamese clinical experts, including obstetricians, pediatricians, neurologists, endocrinologist, geneticist, and family doctors in 7 specialized hospitals at Ho Chi Minh city.Based on the 113 American College of Medical Genetics and Genomics ACMG-recommended genes 16 and suggestions from 104 Vietnamese clinical experts, 540 genes were accounted for in our cohort to evaluate the most common AR and X-linked disorders.Those genes were divided into 3 main groups based on the detrimental effect on quality of life in the population 4,16,31 .Group 1 presented life-threatening disorders, group 2 combined chronic conditions causing cognitive and physical impairment 31,32 , and group 3 had only 3 popular AR genes in Southeast Asia that included GJB2, HBB, and G6PD 14,17 .

Targeted next-generation sequencing and bioinformatics data processing
DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen).Target enrichment was performed with the xGen Custom Hyb Panels (Integrated DNA Technologies, USA).Captured libraries were sequenced with 2 × 150 bp reads on the Nextseq/Novaseq platform (Illumina).Sequence reads were mapped onto the human reference genome hg38 using the BWA tool 33 .The Genome Analysis ToolKit was used for calling variants (SNPs and indels) 33 .Identified variants were annotated using ANNOVAR 34 .The average coverage depth was about 200×, with over 99% of the target regions covered by at least 20 reads.

Variant interpretation
Variants with at least 20× coverage were analyzed using Geneyx (Geneyx Genomex, Israel) 35 and interpreted according to recent ACMG guidelines 36 .Synonymous variants, intronic variants outside of the flanking regions, and variants with a minor allelic frequency (MAF) ≥ 5% in the Genome Aggregation (gnomAD) databases were excluded 37 .Using the Geneyx analysis platform, various in silico prediction programs, including SIFT, PolyPhen-2, MutationTaster, LRT, CADD, and REVEL, were used to analyze missense variants.The analysis of splicing effects was performed with SpliceAI and dbscSNV version 1.1 by AdaBoost and Random Forest, and GERP++ and PhyloP were used to explore nucleotide-specific estimates of evolutionary constraint.All detected pathogenic (P) and likely pathogenic (LP) variants were confirmed by Sanger sequencing.Carriers harboring P/LP variants were classified as genotype-positive.Non-carriers of P/LP variants were considered genotype negative.

Statistics
Continuous variables were expressed as mean ± SD and non-parametric as median (interquartile range).Categorical variables were depicted using numbers (proportions).

Figure 1 .
Figure 1.The overall prevalence of carriers for at least one recessive disease.